In cancers, antigen-specific T cells facilitates antitumor immunity by targeting tumor cells that expresses or presents certain antigens. Therefore, methods for a specific measure of such T cell populations are highly needed in the development process of innovative immune oncology treatments. The immunogenicity of putative antigens is subjected to many intrinsic limitations such as the T-cell repertoire of the patient. There are estimated approximately 108 different T cell clones present in a healthy individual (Ref: Arstila et al., 1999, Science), thus, the individual clone has minute frequency. Hence, it is challenging to establish methods for measuring the frequency of antigen-specific T cells. There are at three common methods for measuring such specific T cell populations, namely the Enzyme-Linked ImmunoSpot (ELISPOT) method, the flow cytometry-based intracellular cytokine staining (ICS) and the MHC multimer staining method also based on flow cytometry. Both the ELISPOT and ICS methods rely on stimulation in vitro prior to measuring secretion of cytokine(s). Regarding the ELISPOT method, most often IFN-γ alone is measured (single cytokine detection) while the ICS assay frequently deals with measuring IFN-γ, TNF-α and IL-2 (multiple cytokine detection) in an effector T cell context. Obviously, only cells secreting cytokine(s) under the applied stimulatory conditions can be detected using both of these methods.
The MHC multimer staining, on contrary, depicts a method for a direct ex vivo measure of the antigen-specific T cells with no stimulation needed. Whereas both ELISPOT and ICS methods relies on the assumption that the activated cells measured are identical to the antigen-specific cells, the MHC multimer method overcomes this limitation. Detection of antigen-specific T cells using the MHC multimer staining method, however, requires knowledge about the patient’ tissue types and the possible exact minimal T cell epitope(s) to screen for.
An advantage of both the ICS and the MHC multimer staining methods is the co-staining of T cell lineage markers and possibly other surface or intracellular markers e.g. involved in maturation, inhibition or activation, enhancing the level of knowledge obtained. By performing multicolor flow cytometry and ICS measurements, we can provide valuable data of the cytokine-producing capabilities of the antigen-specific T cells from patient samples. ICS and MHC multimerer staining methods are always combined with T cell lineage markers and we further offer adding measurement of maturation phenotyping for differentiating the analysis of cytokine production or MHC multimer positive cell populations based on T cell lineage and maturation status.
This advanced and flexible functionality of flow cytometry analysis methods is essential in the development of modern immunotherapies to investigate the observed immune responses upon treatment, while the ELISPOT method is still positioned as the most widely used method for immune monitoring. We offer all three strategies for measuring specific T cell responses at ImmuMap and are happy to help you choosing the most appropriate method(s) for your development study. It is important to stress that these three assays are complementary to each other and each have their strengths as weaknesses.
The ELISPOT method is widely used in laboratories around the world to measure cytokine production by T cells and has obtained status as the golden standard method in this field. It is important, however, to be aware that there are several drawbacks for this method from a methodological perspective. These includes the need for a stimulation period giving a distorted indication of response, usually the limitation for measuring one cytokine at a time, and the lack of cell phenotyping. These factors limit the information provided by ELISPOT, and we therefore prefer offering analysis packages including ELISPOT together with flow cytometry-based measurements.
All our methods for determination of antigen-specific T cell populations can be performed on both cryo-preserved and fresh samples.
ImmuMap provide both ELISPOT and flow cytometry-based assays for immunophenotyping and detection of antigen-specific T cells based on MHC multimer staining or intracellular cytokine staining.